Ischemic stroke, a leading cause of death and disability in the United States, occurs when blood flow to the brain becomes disrupted by an obstruction in a vessel. The oxygen and nutrient deprivation that results initiates a sequence of pathological cellular responses and ultimately destruction of brain tissue, or infarction. Currently, stroke treatment is limited to reperfusion, or the administration of agents that dissolve the obstruction and restore blood flow. A complementary approach to stroke therapy focuses on preventing cell death by rescuing damaged cells from progression to apoptosis. This neuroprotective strategy requires that a therapeutic be delivered to target cells across the blood-brain barrier (BBB) between the bloodstream and the brain. The overall goal of this proposal is to generate and evaluate BBB-permeable therapeutic agents for the treatment of ischemic stroke. As a choice of therapeutic we selected RNA interference technology, a highly- selective and potent gene silencing technique that employs small interfering RNA (siRNA) molecules. We propose to promote the delivery of siRNA using myristoylated poly-arginine peptides (MPAP), novel agents shown by our laboratory to cross the BBB following intravenous administration. Our probes are designed to be detectable by in vivo optical imaging, providing a means to monitor their delivery in model systems of ischemic stroke. As a proof-of-principle that MPAP-siRNA probes are effective neuroprotective agents, we propose to fully characterize and test probes directed against c-src (src) that is implicated in stroke damage in models of ischemic stroke. We propose to test probe uptake and bioactivity in vitro in a variety of brain cells under normal and ischemic conditions. We will then evaluate the biodistribution of MPAP-siRNA probes by in vivo optical imaging. The efficacy of probes will be tested in vivo using a transient middle cerebral artery occlusion model of ischemic stroke in mice. Treatment outcome will be assessed using a clinically-relevant technique for stroke detection, diffusion-weighted magnetic resonance imaging, followed by ex vivo correlation of imaging findings using molecular analysis and histology. We anticipate that MPAP-siRNA probes will prove neuroprotective in models of ischemia. If successful, we intend to demonstrate the flexibility of our delivery module by testing alternative MPAP-siRNA probes directed against additional stroke targets, matrix metalloproteinase 9 (MMP9) and c-jun, in a series of limited pilot studies.